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Abstract:

The present disclosure provides immunoassays and kits for detection or
quantification of an analyte of interest in a test sample that
potentially contains endogenously produced autoantibodies reactive with
the analyte

Claims:

1. A kit for detecting or quantifying an analyte of interest in a test
sample, the kit comprising: a) a solid phase capable of binding
autoantibodies present in the test sample; b) a first antibody that binds
to at least one epitope on the analyte of interest, the first antibody
bound to the solid phase; c) a second antibody that binds to at least one
epitope on the analyte of interest; and d) instructions for detecting or
quantifying the analyte of interest.

2. The kit of claim 1 further comprising a detectable label conjugated to
the second antibody.

4. The kit of claim 3 wherein the detectable label is an acridinium
compound.

5. The kit of claim 4, wherein the acridinium compound is an
acridinium-9-carboxamide having a structure according to formula I:
##STR00011## wherein R1 and R2 are each independently selected
from the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl,
sulfoalkyl, carboxyalkyl and oxoalkyl, and wherein R3 through
R15 are each independently selected from the group consisting of:
hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl,
alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo,
sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup.Θ is an anion.

6. The kit of claim 4 wherein the acridinium compound IS an
acridinium-9-carboxylate aryl ester having a structure according to
formula II: ##STR00012## wherein R1 is an alkyl, alkenyl, alkynyl,
aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and wherein
R3 through R15 are each independently selected from the group
consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino,
amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano,
sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if present,
X.sup.Θ is an anion.

7. The kit of claim 4 further comprising a basic solution.

8. The kit of claim 7 wherein the basic solution is a solution having a
pH of at least about 10.

9. The kit of claim 4 further comprising a hydrogen peroxide source.

10. The kit of claim 9 wherein the hydrogen peroxide source comprises a
buffer or a solution containing hydrogen peroxide.

13. The kit of claim 1, wherein the solid phase is selected from the
group consisting of a magnetic particle, a bead, a test tube, a
microtiter plate, a cuvette, a membrane, a scaffolding molecule, a quartz
crystal, a film, a filter paper, a disc and a chip.

14. The kit of claim 1, wherein the first antibody and the second
antibody each bind to an epitope on an analyte of interest selected from
the group consisting of cardiac troponin, thyroid stimulating hormone
(TSH), beta human chorionic gonadotropin (beta-HCG); myeloperoxidase
(MPO), prostate specific antigen (PSA), human B-type natriuretic peptide
(BNP), myosin light chain 2, myosin-6 and myosin-7.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of allowed U.S. patent
application Ser. No. 12/630,697, hereby incorporated in its entirety by
reference.

TECHNICAL FIELD

[0002] The present disclosure relates to immunoassays and kits for
detecting an analyte of interest in a test sample, and in particular to
methods and kits for detecting an analyte in a human test sample that may
contain endogenous anti-analyte antibodies.

BACKGROUND

[0003] Immunoassay techniques have been known for the last few decades and
are now commonly used in medicine for a wide variety of diagnostic
purposes to detect target analytes in a biological sample. Immunoassays
exploit the highly specific binding of an antibody to its corresponding
antigen, wherein the antigen is the target analyte. Typically,
quantification of either the antibody or antigen is achieved through some
form of labeling such as radio- or fluorescence-labeling. Sandwich
immunoassays involve binding the target analyte in the sample to the
antibody site (which is frequently bound to a solid support), binding
labeled antibody to the captured analyte, and then measuring the amount
of bound labeled antibody, wherein the label generates a signal
proportional to the concentration of the target analyte inasmuch as
labeled antibody does not bind unless the analyte is present in the
sample.

[0004] A problem with this general approach is that many patients have
circulating endogenous antibodies, or "autoantibodies" against an analyte
of clinical interest. For example, autoantibodies have been described for
cardiac troponin, myeloperoxidase (MPO), prostate specific antigen (PSA),
and thyroid stimulating hormone (TSH), and other clinically significant
analytes. Autoantibodies create interference in typical sandwich
immunoassays that are composed of two or more analyte-specific
antibodies. For example, cardiac troponin-reactive autoantibodies may
interfere with the measurement of cTnI using conventional
midfragment-specific immunoassays. Thus, interference from autoantibodies
can produce erroneous results, particularly near the cut-off values
established for clinical diagnoses, and increases the risk of false
negative diagnostic results and the risk that individuals will not obtain
a timely diagnosis.

[0005] One approach to addressing this problem is to choose
analyte-specific antibodies that bind to specific epitopes distinct from
the analyte epitopes that react with the autoantibodies. Following this
general approach, efforts have focused on exploring the use of thousands
of different combinations of two, three and even four analyte-specific
antibodies to avoid interference from autoantibodies. However, this
effort has been largely unsuccessful. It is now evident that
autoantibodies against complex protein analytes are likely to be
polyclonal within a particular sample, and may be even more diverse among
samples from different individuals. Interference from diverse polyclonal
autoantibodies may explain the observation that as little as 25% or even
less of an analyte protein sequence binds to analyte-specific antibodies,
which may in turn explain the lack of success using this approach.

[0006] A need exists in the art for new immunoassay methods that
compensate for interference by autoantibodies in a sample, and in
particular for such methods that do so without involving redesign of the
analyte detection or capture antibodies.

SUMMARY

[0007] In one aspect, the present disclosure relates to an immunoassay for
detecting an analyte of interest in a test sample, the immunoassay
comprising the steps of:

[0008] (a) contacting a test sample suspected of containing an analyte of
interest with a first antibody that binds to at least one epitope on the
analyte of interest to form a first antibody-analyte complex, wherein the
first antibody is immobilized on a solid phase, and further wherein at
least one autoantibody in the test sample binds to at least one epitope
on the analyte of interest to form an autoantibody-analyte complex,
wherein said autoantibody binds to the solid phase;

[0009] (b) contacting said mixture comprising a first antibody-analyte
complex and an autoantibody-analyte complex with a second antibody to
form a measurable assembly comprising a first antibody-analyte-second
antibody complex and an autoantibody-analyte-second antibody complex;
wherein the second antibody binds to at least one epitope on the analyte
of interest, and further wherein, an optical, electrical, or
change-of-state signal of the assembly is measured.

[0010] In the above immunoassay, the second antibody can be conjugated to
a detectable label, wherein the detectable label is an enzyme,
oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence
quencher, chemiluminescence quencher, or biotin.

[0012] In the above immunoassay, the electrical signal can be measured as
an analyte concentration dependent change in current, resistance,
potential, mass to charge ratio, or ion count.

[0013] In the above immunoassay, the change-of-state signal can be
measured as an analyte concentration dependent change in size,
solubility, mass, or resonance.

[0014] In another aspect, the present disclosure relates to an immunoassay
for detecting an analyte of interest in a test sample, the immunoassay
comprising the steps of:

[0015] (a) contacting a test sample suspected of containing an analyte of
interest with a first antibody that binds to at least one epitope on the
analyte of interest to form a first antibody-analyte complex, wherein the
first antibody is immobilized on a solid phase, and further wherein at
least one autoantibody in the test sample binds to at least one epitope
on the analyte of interest to form an autoantibody-analyte complex,
wherein said autoantibody binds to the solid phase;

[0016] (b) contacting said mixture comprising a first antibody-analyte
complex and an autoantibody-analyte complex, with a second antibody that
has been conjugated to a detectable label to form a first
antibody-analyte-second antibody complex and an
autoantibody-analyte-second antibody complex; wherein the second antibody
binds to at least one epitope on the analyte of interest and further
wherein, the detectable label is at least one acridinium compound;

[0017] (c) generating or providing a source of hydrogen peroxide to the
mixture of step (b);

[0018] (d) adding a basic solution to the mixture of step (c) to generate
a light signal; and

[0019] (e) measuring the light signal generated by or emitted in step (d)
and detecting the analyte of interest in the test sample.

[0021] In the above immunoassay, the test sample can be whole blood,
serum, or plasma. In the above immunoassay, any acridinium compound can
be used. For example, the acridinium compound can be an
acridinium-9-carboxamide having a structure according to formula I:

##STR00001##

[0022] wherein R1 and R2 are each independently selected from the group
consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,
carboxyalkyl and oxoalkyl, and

[0029] In the above immunoassay, the solid phase can be selected from the
group consisting of a magnetic particle, bead, test tube, microtiter
plate, cuvette, membrane, a scaffolding molecule, quartz crystal, film,
filter paper, disc and chip.

[0030] In the above immunoassay, the first antibody can be selected from
the group consisting of a polyclonal antibody, a monoclonal antibody, a
chimeric antibody, a human antibody, and an affinity maturated antibody.

[0031] In the above immunoassay the second antibody can be selected from
the group consisting of a polyclonal antibody, a monoclonal antibody, a
chimeric antibody, a human antibody, and an affinity maturated antibody.

[0032] In the above immunoassay the hydrogen peroxide can be provided by
adding a buffer or a solution containing hydrogen peroxide.

[0034] In the above immunoassay, the basic solution can be a solution
having a pH of at least about 10.

[0035] Optionally, the above immunoassay may further comprise the step of
quantifying the amount of the analyte of interest in the test sample by
relating the amount of light signal in step (e) to the amount of the
analyte of interest in the test sample either by use of a standard curve
for the analyte of interest or by comparison to a reference standard. The
immunoassay may be adapted for use in an automated system or
semi-automated system.

[0036] In another aspect, the present disclosure relates to a kit for
detecting or quantifying an analyte of interest in a test sample, the kit
comprising a solid phase capable of binding autoantibodies present in the
test sample; a first antibody that binds to at least one epitope on the
analyte of interest, the first antibody bound to the solid phase; a
second antibody that binds to at least one epitope on the analyte of
interest; and instructions for detecting or quantifying the analyte of
interest.

[0037] In the above kit, a detectable label can be conjugated to the
second antibody. The detectable label can be an enzyme, oligonucleotide,
nanoparticle chemiluminophore, fluorophore, fluorescence quencher,
chemiluminescence quencher, or biotin. In certain embodiments, the
detectable label is an acridinium compound. The acridinium compound can
be an acridinium-9-carboxamide having a structure according to formula I:

##STR00003##

[0038] wherein R1 and R2 are each independently selected from the group
consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl,
carboxyalkyl and oxoalkyl, and

[0039] wherein R3 through R15 are each independently selected from the
group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl,
amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro,
cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and optionally, if
present, X.sup.Θ is an anion. Alternatively, the acridinium
compound can be an acridinium-9-carboxylate aryl ester having a structure
according to formula II:

##STR00004##

[0040] wherein R1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl,
sulfoalkyl, carboxyalkyl and oxoalkyl; and

[0044] In the above it, the solid phase can be selected from the group
consisting of a magnetic particle, a bead, a test tube, a microtiter
plate, a cuvette, a membrane, a scaffolding molecule, a quartz crystal, a
film, a filter paper, a disc and a chip.

[0045] In the above it, the first antibody and the second antibody can
each bind to an epitope on an analyte of interest selected from the group
consisting of cardiac troponin, thyroid stimulating hormone (TSH), beta
human chorionic gonadotropin (beta-HCG); myeloperoxidase (MPO), prostate
specific antigen (PSA), human B-type natriuretic peptide (BNP), myosin
light chain 2, myosin-6 and myosin-7.

[0046] In another aspect, the present disclosure relates to an
immunodetection composition including a first detection complex
comprising a first antibody reactive with an analyte of interest and
bound to a solid phase, the analyte of interest, and a second antibody
reactive with the analyte of interest, wherein the second antibody has a
detectable label, and a second detection complex comprising an
autoantibody reactive with the analyte of interest and bound to the solid
phase, the analyte of interest, and the second antibody, wherein the
first and second complexes generate a measurable optical, electrical, or
change-of-state signal from the detectable label. In the above
immunodetection composition, the first detection complex can be bound to
the second detection complex on the solid phase.

[0048] FIG. 2 shows a schematic diagram of an immunoassay reaction
sequence in which autoantibodies reactive with a target analyte and
autoantibodies unrelated to the target analyte are bound to a solid phase
bearing an exogenous capture antibody;

[0049] FIG. 3 shows a graph of dose-response of human IgG captured on
magnetic microparticles and conjugated to an analyte-specific IgG; and

[0050] FIG. 4 shows a graph of a serial dilution of IgG from human serum
captured on magnetic microparticles and conjugated to an analyte specific
IgG.

DETAILED DESCRIPTION

[0051] The present disclosure relates to immunoassay methods and kits for
detecting an analyte of interest in a test sample, and more particularly
to methods and kits for detecting an analyte in a human test sample that
may contain endogenous anti-analyte antibodies. Specifically, the
inventors have discovered an alternative approach to address the problem
of autoantibodies in immunoassay detection of clinically significant
analytes in a sample, in which a solid phase that bears an
analyte-specific antibody is also capable of binding autoantibodies
against the analyte that may be present in the sample. This assay
approach compensates for the presence of autoantibodies in the sample
without redesign of the analyte-specific detection antibodies or the
capture antibodies, does not require use of an extra anti-human IgG
detection conjugate, and avoids the need of a second assay to identify
problematic samples.

A. DEFINITIONS

[0052] Section headings as used in this section and the entire disclosure
herein are not intended to be limiting.

[0053] As used herein, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise. For the
recitation of numeric ranges herein, each intervening number there
between with the same degree of precision is explicitly contemplated. For
example, for the range 6-9, the numbers 7 and 8 are contemplated in
addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly
contemplated.

[0054] a) Acyl (and Other Chemical Structural Group Definitions)

[0055] As used herein, the term "acyl" refers to a --C(O)Ra group
where Ra is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or
phenylalkyl. Representative examples of acyl include, but are not limited
to, formyl, acetyl, cylcohexylcarbonyl, cyclohexylmethylcarbonyl,
benzoyl, benzylcarbonyl and the like.

[0056] As used herein, the term "alkenyl" means a straight or branched
chain hydrocarbon containing from 2 to 10 carbons and containing at least
one carbon-carbon double bond formed by the removal of two hydrogens.
Representative examples of alkenyl include, but are not limited to,
ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl,
5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0058] As used herein, the term "alkyl radical" means any of a series of
univalent groups of the general formula CnH2n+1 derived from
straight or branched chain hydrocarbons.

[0059] As used herein, the term "alkoxy" means an alkyl group, as defined
herein, appended to the parent molecular moiety through an oxygen atom.
Representative examples of alkoxy include, but are not limited to,
methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and
hexyloxy.

[0060] As used herein, the term "alkynyl" means a straight or branched
chain hydrocarbon group containing from 2 to 10 carbon atoms and
containing at least one carbon-carbon triple bond. Representative
examples of alkynyl include, but are not limited, to acetylenyl,
1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

[0061] As used herein, the term "amido" refers to an ammo group attached
to the parent molecular moiety through a carbonyl group (wherein the term
"carbonyl group" refers to a --C(O)-- group).

[0062] As used herein, the term "amino" means --NRbRc, wherein
Rb, and Rc, are independently selected from the group
consisting of hydrogen, alkyl and alkylcarbonyl.

[0063] As used herein, the term "aralkyl" means an aryl group appended to
the parent molecular moiety through an alkyl group, as defined herein.
Representative examples of aryl alkyl include, but are not limited to,
benzyl, 2-phenylethyl, 3-phenylpropyl, and 2-naphth-2-ylethyl.

[0064] As used herein, the term "aryl" means a phenyl group, or a bicyclic
or tricyclic fused ring system wherein one or more of the fused rings is
a phenyl group. Bicyclic fused ring systems are exemplified by a phenyl
group fused to a cycloalkenyl group, a cycloalkyl group, or another
phenyl group. Tricyclic fused ring systems are exemplified by a bicyclic
fused ring system fused to a cycloalkenyl group, a cycloalkyl group, as
defined herein or another phenyl group. Representative examples of aryl
include, but are not limited to, anthracenyl, azulenyl, fluorenyl,
indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. The aryl
groups of the present disclosure can be optionally substituted with one-,
two, three, four, or five substituents independently selected from the
group consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0065] As used herein, the term "carboxy" or "carboxyl" refers to
--CO2H or --CO2.

[0066] As used herein, the term "carboxyalkyl" refers to a
--(CH2)nCO2H or --(CH2)nCO2-- group where n
is from 1 to 10.

[0067] As used herein, the term "cyano" means a --CN group.

[0068] As used herein, the term "cycloalkenyl" refers to a non-aromatic
cyclic or bicyclic ring system having from three to ten carbon atoms and
one to three rings, wherein each five-membered ring has one double bond,
each six-membered ring has one or two double bonds, each seven- and
eight-membered ring has one to three double bonds, and each nine-to
ten-membered ring has one to four double bonds. Representative examples
of cycloalkenyl groups include cyclohexenyl, octahydronaphthalenyl,
norbornylenyl, and the like. The cycloalkenyl groups can be optionally
substituted with one, two, three, four, or five substituents
independently selected from the group consisting of alkoxy, alkyl,
carboxyl, halo, and hydroxyl.

[0069] As used herein, the term "cycloalkyl" refers to a saturated
monocyclic, bicyclic, or tricyclic hydrocarbon ring system having three
to twelve carbon atoms. Representative examples of cycloalkyl groups
include cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl, adamantyl, and
the like. The cycloalkyl groups of the present disclosure can be
optionally substituted with one, two, three, four, or five substituents
independently selected from the group consisting of alkoxy, alkyl,
carboxyl, halo, and hydroxyl.

[0070] As used herein, the term "cycloalkylalkyl" means a --RdRe
group where Rd is an alkylene group and Re is cycloalkyl group.
A representative example of a cycloalkylalkyl group is cyclohexylmethyl
and the like.

[0071] As used herein, the term "halogen" means a --Cl, --Br, --I or --F;
the term "halide" means a binary compound, of which one part is a halogen
atom and the other part is an element or radical that is less
electronegative than the halogen, e.g., an alkyl radical.

[0072] As used herein, the term "hydroxyl" means an --OH group.

[0073] As used herein, the term "nitro" means a --NO2 group.

[0074] As used herein, the term "oxoalkyl" refers to
--(CH2)nC(O)Ra, where Ra is hydrogen, alkyl,
cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl and where n is from 1
to 10.

[0075] As used herein, the term "phenylalkyl" means an alkyl group which
is substituted by a phenyl group.

[0076] As used herein, the term "sulfo" means a --SO3H group.

[0077] As used herein, the term "sulfoalkyl" refers to a
--(CH2)nSO3H or --(CH2)nSO3-- group where n
is from 1 to 10.

[0081] As used herein, the term "antibody" refers to a protein consisting
of one or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes, and encompasses polyclonal
antibodies, monoclonal antibodies, and fragments thereof, as well as
molecules engineered from immunoglobulin gene sequences. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma, delta,
epsilon and mu constant region genes, as well as myriad immunoglobulin
variable region genes. Light chains are classified as either kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or
epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,
IgD and IgE, respectively.

[0082] d) Hydrogen Peroxide Generating Enzyme

[0083] As used herein, the term "hydrogen peroxide generating enzyme"
refers to an enzyme that is capable of producing as a reaction product
the chemical compound having the molecular formula H2O2 i.e.
hydrogen peroxide. Non-limiting examples of hydrogen peroxide generating
enzymes are listed below in Table 1.

[0085] As used herein, the phrase "autoantibody" refers to an antibody
that binds to an analyte that is endogenously produced in the subject in
which the antibody is produced.

[0086] e) Antibody-Analyte Complex

[0087] As used herein, the phrase "antibody-analyte complex" refers to a
combination of an antibody and an antigen, in which the antigen is an
analyte of interest, and the antibody and antigen are bound by specific,
noncovalent interactions between an antigen-combining site on the
antibody and an antigen epitope. The antigen may be a protein or other
molecule. The term "autoantibody-analyte complex" encompasses an
antibody-analyte complex in which the antibody is an antibody that binds
to an analyte that is endogenously produced in the subject in which the
antibody is produced.

[0088] g) Detectable Label

[0089] As used herein the term "detectable label" refers to any moiety
that generates a measurable signal via optical, electrical, or other
physical indication of a change of state of a molecule or molecules
coupled to the moiety. Such physical indicators encompass spectroscopic,
photochemical, biochemical, immunochemical, electromagnetic,
radiochemical, and chemical means, such as but not limited to
fluorescence, chemifluorescence, chemiluminescence, and the like.
Preferred detectable labels include acridinium compounds such as an
acridinium-9-carboximide having a structure according to Formula I as set
forth in section B herein below, and an acridinium-9-carboxylate aryl
ester having a structure according to Formula II as also set forth in
section B herein below.

[0090] h) Subject

[0091] As used herein, the terms "subject" and "patient" are used
interchangeably irrespective of whether the subject has or is currently
undergoing any form of treatment. As used herein, the terms "subject" and
"subjects" refer to any vertebrate, including, but not limited to, a
mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep,
hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for
example, a monkey, such as a cynomolgous monkey, chimpanzee, etc) and a
human). Preferably, the subject is a human.

[0092] i) Test Sample

[0093] As used herein, the term "test sample" generally refers to a
biological material being tested for and/or suspected of containing an
analyte of interest and which may also include autoantibodies to the
analyte of interest. The biological material may be derived from any
biological source but preferably is a biological fluid likely to contain
the analyte of interest. Examples of biological materials include, but
are not limited to, stool, whole blood, serum, plasma, red blood cells,
platelets, interstitial fluid, saliva, ocular lens fluid, cerebral spinal
fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum, synovial
fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen,
soil, etc. The test sample may be used directly as obtained from the
biological source or following a pretreatment to modify the character of
the sample. For example, such pretreatment may include preparing plasma
from blood, diluting viscous fluids and so forth. Methods of pretreatment
may also involve filtration, precipitation, dilution, distillation,
mixing, concentration, inactivation of interfering components, the
addition of reagents, lysing, etc. If such methods of pretreatment are
employed with respect to the test sample, such pretreatment methods are
such that the analyte of interest remains in the test sample at a
concentration proportional to that in an untreated test sample (e.g.,
namely, a test sample that is not subjected to any such pretreatment
method/sf).

B. IMMUNOASSAY FOR DETECTING AN ANALYTE OF INTEREST IN A TEST SAMPLE THAT
MAY CONTAIN AUTOANTIBODIES

[0094] The present disclosure relates to an immunoassay for detecting an
analyte of interest in a test sample in which autoantibodies against the
analyte of interest mayor may not be present. Examples of analytes of
interest for which autoantibodies have been described include but are not
limited to cardiac troponin, myeloperoxidase (MPO), prostate specific
antigen (PSA), and thyroid stimulating hormone (TSH). It will be
understood that the immunoassays described herein are also applicable to
the detection of any other analyte of interest for which autoantibodies
not yet described create the risk of interference for immunodetection of
the analyte.

[0095] The immunoassay of the present disclosure involves obtaining a test
sample from a subject and then detecting the presence of an analyte of
interest using immunodetection while compensating for the presence of any
autoantibodies against the analyte that may be present in the sample.
This is achieved in part by providing a solid phase, which can be a solid
support, on which a first, capture antibody is immobilized, and which
also during the course of the immunoassay binds any autoantibody that may
be present in the sample.

Immunoassay Methods

[0096] The immunoassay methods of the present disclosure can be carried
out in any of a wide variety of formats. A general review of immunoassays
is available in METHODS IN CELL BIOLOGY VOLUME 37: ANTIBODIES IN CELL
BIOLOGY, Asai, ed. Academic Press, Inc. New York (1993), and BASIC AND
CLINICAL IMMUNOLOGY 7TH EDITION, Stites & Ten, eds. (1991), which are
herein incorporated by reference in its entirety. FIG. 1 is a schematic
diagram of a typical heterogeneous sandwich immunoassay employing a solid
phase (as a solid support) to which is bound a first (capture) antibody
reactive with at least one epitope on the analyte of interest. A second
(detection) antibody is also reactive with at least one epitope on the
analyte of interest. As is shown in FIG. 1, the second antibody may be
conjugated to a detectable label (as indicated by the starburst icon)
that provides a signal that is measured after the detection antibody
binds to the captured analyte. When a test sample containing the analyte
of interest contacts the first antibody, the first antibody captures the
analyte. The analyte is contacted with the second antibody resulting in
the formation of an immunodetection complex consisting of the first
antibody, analyte and second antibody, and the complex is bound to the
solid phase. The signal generated by the second (detection) antibody is
proportional to the concentration of the analyte as determined by the
rate of formation (k1) of the immunodetection complex versus the
rate of dissociation of the immunodetection complex (k2). As can be
inferred from FIG. 1, autoantibodies, which if present are unpredictable
as to exactly where on an analyte they will bind, can substantially
interfere with binding of the first and/or second antibody, and thus with
the resulting signal.

[0097] In contrast to an Immunoassay format as described and illustrated
in FIG. 1, immunoassays according to the present disclosure employ a
solid phase that bears a first (capture) antibody as in FIG. 1, but also
is capable of binding any autoantibodies that may be present in the test
sample. FIG. 2 is a schematic diagram of an immunoassay format according
to the present disclosure, in which the test sample contains multiple
autoantibodies as shown, each reactive with at least one different
epitope on the analyte of interest. The test sample may also contain
autoantibodies that are unrelated to the analyte (not shown). As shown in
FIG. 2, the solid phase captures the analyte via binding of the analyte
to the first antibody, but also by directly binding autoantibodies that
are reactive with the analyte (as well as any unrelated autoantibodies
that are not reactive with the analyte). The result under appropriate
conditions is formation of an immunodetection complex that includes a
first antibody-analyte-second antibody complex, and an
autoantibody-analyte-second antibody complex, which generates a stronger
signal than that produced by the immunodetection complex shown in FIG. 1.
In the immunoassay of the present disclosure and as shown in FIG. 2, the
signal generated by the second (detection) antibody remains proportional
to the concentration of the analyte as determined by the rate of
formation (k3) of the new immunodetection complex versus the rate of
dissociation of the new immunodetection complex (k4). In test
samples containing no autoantibodies reactive with the analyte,
autoantibodies unrelated to the analyte are bound by the solid support,
but hey do not bind any of the analyte of interest and the signal
indicative of the analyte is unaffected.

[0098] Thus, according to the present disclosure, an immunoassay of the
present disclosure to detect the presence of an analyte of interest is a
heterogeneous assay employing a solid phase which can be a solid support.
The immunoassay can be performed for example by immobilizing a first
antibody on the solid phase, wherein the first antibody is an exogenous
capture antibody, i.e. an exogenous antibody that is reactive with at
least one epitope on the analyte of interest. The solid phase is also
capable of binding any endogenous autoantibodies that may be present in
the sample. Under conditions sufficient for specific binding of the first
antibody to the analyte of interest, the test sample suspected of
containing the analyte of interest, and which mayor may not contain
autoantibodies, is contacted with the first (capture) antibody, thus
forming a first antibody-analyte complex. In the case of a test sample
containing at least one autoantibody against the analyte, the
autoantibody binds to the solid phase and also can bind to at least one
epitope on the analyte to form an autoantibody-analyte complex. A mixture
thus formed of the first antibody-analyte complex and the
autoantibody-analyte complex is contacted with a second, detection
antibody that binds to at least one epitope on the analyte of interest.
This step is carried out under conditions sufficient for specific binding
of the second antibody to any of the analyte of interest that is present
in the test sample. The second antibody binds to the analyte to form an
immunodetection complex which forms a measurable assembly including the
first antibody-analyte-second antibody complex and the
autoantibody-analyte-second antibody complex. By "measurable assembly" is
meant a configuration of molecules that when formed generates a signal
susceptible to physical detection and/or quantification. In certain
embodiments for example, the second antibody may be labeled with a
detectable label. Depending on the detection approach used, an optical,
electrical, or change-of-state signal of the assembly is measured.

[0099] Although the immunoassay is described above as including a sequence
of steps for illustrative purposes, the test sample may be contacted with
the first (capture) antibody and the second (detection) antibody
simultaneously or sequentially, in any order. Regardless of the order of
contact, if autoantibodies are present in the sample, the autoantibodies
bind directly to the solid phase. Only those autoantibodies that are
reactive with the analyte of interest form part of the immunodetection
complex that contains the analyte bound to the first, capture antibody,
any autoantibody reactive with the analyte, and the second, detection
antibody.

[0100] In one format of a sandwich immunoassay according to the present
disclosure, detecting comprises detecting a signal from the solid
phase-affixed immunodetection complex which is a measurable assembly
including a first antibody-analyte-second antibody complex and an
autoantibody-analyte-second antibody complex. In one embodiment, the
immunodetection complex is separated from the solid phase, typically by
washing, and the signal from the bound label is detected. In another
format of a sandwich immunoassay according to the present disclosure, the
immunodetection complex remains a solid phase-affixed complex, which is
then detected.

Antibodies

[0101] In the immunoassays according to the present disclosure, the first
antibody can be a polyclonal antibody, a monoclonal antibody, a chimeric
antibody, a human antibody, an affinity maturated antibody or an antibody
fragment. Similarly, the second antibody can be a polyclonal antibody, a
monoclonal antibody, a chimeric antibody, a human antibody, an affinity
maturated antibody or an antibody fragment.

[0102] While monoclonal antibodies are highly specific to the
analyte/antigen, a polyclonal antibody can preferably be used as the
capture (first) antibody to immobilize as much of the analyte/antigen as
possible. A monoclonal antibody with inherently higher binding
specificity for the analyte/antigen may then preferably be used as the
detection (second) antibody. In any case, the capture and detection
antibodies preferably recognize two non-overlapping epitopes on the
analyte to avoid blockage of, or interference by the capture antibody
with the epitope recognized by the detection antibody. Preferably the
capture and detection antibodies are capable of binding simultaneously to
different epitopes on the analyte, each without interfering with the
binding of the other.

[0103] Polyclonal antibodies are raised by injecting (e.g., subcutaneous
or intramuscular injection) an immunogen into a suitable non-human mammal
(e.g., a mouse or a rabbit). Generally, the immunogen should induce
production of high titers of antibody with relatively high affinity for
the target antigen.

[0104] If desired, the antigen may be conjugated to a carrier protein by
conjugation techniques that are well known in the art. Commonly used
carriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine
serum albumin (BSA), and tetanus toxoid. The conjugate is then used to
immunize the animal.

[0105] The antibodies are then obtained from blood samples taken from the
animal. The techniques used to produce polyclonal antibodies are
extensively described in the literature (see, e.g., Methods of
Enzymology, "Production of Antisera With Small Doses of Immunogen:
Multiple Intradermal Injections," Langone, et al. eds. (Acad. Press,
1981)). Polyclonal antibodies produced by the animals can be further
purified, for example, by binding to and elution from a matrix to which
the target antigen is bound. Those of skill in the art will know of
various techniques common in the immunology arts for purification and/or
concentration of polyclonal, as well as monoclonal, antibodies (see,
e.g., Coligan, et al. (1991) Unit 9, Current Protocols in Immunology,
Wiley Interscience).

[0106] For many applications, monoclonal antibodies (mAbs) are preferred.
The general method used for production of hybridomas secreting mAbs is
well known (Kohler and Milstein (1975) Nature, 256:495). Briefly, as
described by Kohler and Milstein, the technique entailed isolating
lymphocytes from regional draining lymph nodes of five separate cancer
patients with either melanoma, teratocarcinoma or cancer of the cervix,
glioma or lung, (where samples were obtained from surgical specimens),
pooling the cells, and fusing the cells with SHFP-1. Hybridomas were
screened for production of antibody that bound to cancer cell lines.
Confirmation of specificity among mAbs can be accomplished using routine
screening techniques (such as the enzyme-linked immunosorbent assay, or
"ELISA") to determine the elementary reaction pattern of the mAb of
interest.

[0107] As used herein, the term "antibody" encompasses antigen-binding
antibody fragments, e.g., single chain antibodies (scFv or others), which
can be produced/selected using phage display technology. The ability to
express antibody fragments on the surface of viruses that infect bacteria
(bacteriophage or phage) makes it possible to isolate a single binding
antibody fragment, e.g., from a library of greater than 1010
nonbinding clones. To express antibody fragments on the surface of phage
(phage display), an antibody fragment gene is inserted into the gene
encoding a phage surface protein (e.g., pIII) and the antibody
fragment-pIII fusion protein is displayed on the phage surface
(McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991)
Nucleic Acids Res. 19: 4133-4137).

[0108] Since the antibody fragments on the surface of the phage are
functional, phage-bearing antigen-binding antibody fragments can be
separated from non-binding phage by antigen affinity chromatography
(McCafferty et al. (1990) Nature, 348: 552-554). Depending on the
affinity of the antibody fragment, enrichment factors of
20-fold-1,000,000-fold are obtained for a single round of affinity
selection. By infecting bacteria with the eluted phage, however, more
phage can be grown and subjected to another round of selection. In this
way, an enrichment of 1000-fold in one round can become 1,000,000-fold in
two rounds of selection (McCafferty et al. (1990) Nature, 348: 552-554).
Thus, even when enrichments are low (Marks et al. (1991) J. Mol. Biol.
222: 581-597), multiple rounds of affinity selection can lead to the
isolation of rare phage. Since selection of the phage antibody library on
antigen results in enrichment, the majority of clones bind antigen after
as few as three to four rounds of selection. Thus only a relatively small
number of clones (several hundred) need to be analyzed for binding to
antigen.

[0109] Human antibodies can be produced without prior immunization by
displaying very large and diverse V-gene repertoires on phage (Marks et
al. (1991) J. Mol. Biol. 222: 581-597). In one embodiment, natural VH and
VL repertoires present in human peripheral blood lymphocytes are isolated
from unimmunized donors by PCR. The V-gene repertoires can be spliced
together at random using PCR to create a scFv gene repertoire which can
be cloned into a phage vector to create a library of 30 million phage
antibodies (Id.). From a single "naive" phage antibody library, binding
antibody fragments have been isolated against more than 17 different
antigens, including haptens, polysaccharides, and proteins (Marks et al.
(1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993). Bio/Technology.
10: 779-783; Griffiths et al. (1993) EMBO J. 12: 725-734; Clackson et al.
(1991) Nature. 352: 624-628). Antibodies have been produced against self
proteins, including human thyroglobulin, immunoglobulin, tumor necrosis
factor, and CEA (Griffiths et al. (1993) EMBO J. 12: 725-734). The
antibody fragments are highly specific for the antigen used for selection
and have affinities in the 1 nM to 100 nM range (Marks et al. (1991) J.
Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12: 725-734).
Larger phage antibody libraries result in the isolation of more
antibodies of higher binding affinity to a greater proportion of
antigens.

[0110] As those of skill in the art readily appreciate, antibodies can be
prepared by any of a number of commercial services (e.g., Berkeley
Antibody Laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).

Solid Phase

[0111] The solid phase can be any suitable material with sufficient
surface affinity to bind a capture antibody and autoantibodies present in
the test sample. The solid phase can take any of a number of forms, such
as a magnetic particle, bead, test tube, microtiter plate, cuvette,
membrane, a scaffolding molecule, quartz crystal, film, filter paper,
disc or a chip. Useful solid phase materials include: natural polymeric
carbohydrates and their synthetically modified, crosslinked, or
substituted derivatives, such as agar, agarose, cross-linked alginic
acid, substituted and cross-linked guar gums, cellulose esters,
especially with nitric acid and carboxylic acids, mixed cellulose esters,
and cellulose ethers; natural polymers containing nitrogen, such as
proteins and derivatives, including cross-linked or modified gelatins;
natural hydrocarbon polymers, such as latex and rubber; synthetic
polymers, such as vinyl polymers, including polyethylene, polypropylene,
polystyrene, polyvinylchloride, polyvinylacetate and its partially
hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers
and terpolymers of the above polycondensates, such as polyesters,
polyamides, and other polymers, such as polyurethanes or polyepoxides;
inorganic materials such as sulfates or carbonates of alkaline earth
metals and magnesium, including barium sulfate, calcium sulfate, calcium
carbonate, silicates of alkali and alkaline earth metals, aluminum and
magnesium; and aluminum or silicon oxides or hydrates, such as clays,
alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may
be used as filters with the above polymeric materials); and mixtures or
copolymers of the above classes, such as graft copolymers obtained by
initializing polymerization of synthetic polymers on a pre-existing
natural polymer. All of these materials may be used in suitable shapes,
such as films, sheets, tubes, particulates, or plates, or they may be
coated onto, bonded, or laminated to appropriate inert carriers, such as
paper, glass, plastic films, fabrics, or the like. Nitrocellulose has
excellent absorption and adsorption qualities for a wide variety of
reagents including monoclonal antibodies. Nylon also possesses similar
characteristics and also is suitable.

[0112] Alternatively, the solid phase can constitute microparticles.
Microparticles useful in the present disclosure can be selected by one
skilled in the art from any suitable type of particulate material and
include those composed of polystyrene, polymethylacrylate, polypropylene,
latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or
similar materials. Further, the microparticles can be magnetic or
paramagnetic microparticles, so as to facilitate manipulation of the
microparticle within a magnetic field. In an exemplary embodiment the
microparticles are carboxylated magnetic microparticles.

[0113] Microparticles can be suspended in the mixture of soluble reagents
and test sample or can be retained and immobilized by a support material.
In the latter case, the microparticles on or in the support material are
not capable of substantial movement to positions elsewhere within the
support material. Alternatively, the microparticles can be separated from
suspension in the mixture of soluble reagents and test sample by
sedimentation or centrifugation. When the microparticles are magnetic or
paramagnetic the microparticles can be separated from suspension in the
mixture of soluble reagents and test sample by a magnetic field.

[0114] The methods of the present disclosure can be adapted for use in
systems that utilize microparticle technology including automated and
semi-automated systems wherein the solid phase comprises a microparticle.
Such systems include those described in pending U.S. application Ser. No.
425,651 and U.S. Pat. No. 5,089,424, which correspond to published EPO
App. Nos. EP 0 425 633 and EP 0 424 634, respectively, and U.S. Pat. No.
5,006,309.

[0115] In particular embodiments, the solid phase includes one or more
electrodes. Capture antibodies can be affixed, directly or indirectly, to
the electrode(s). In one embodiment, for example, capture antibodies can
be affixed to magnetic or paramagnetic microp articles, which are then
positioned in the vicinity of the electrode surface using a magnet.
Systems in which one or more electrodes serve as the solid phase are
useful where detection is based on electrochemical interactions.
Exemplary systems of this type are described, for example, in U.S. Pat.
No. 6,887,714 (issued May 3, 2005). The basic method is described further
below with respect to electrochemical detection.

[0116] The capture antibody can be attached to the solid phase by
adsorption, where it is retained by hydrophobic forces. Alternatively,
the surface of the solid phase can be activated by chemical processes
that cause covalent linkage of the capture antibody to the support.

[0117] To change or enhance the intrinsic charge of the solid phase, a
charged substance can be coated directly onto the solid phase. Ion
capture procedures for immobilizing an immobilizable reaction complex
with a negatively charged polymer, described in U.S. application Ser. No.
150,278, corresponding to EP Publication No. 0326100, and U.S.
application Ser. No. 375,029 (EP Publication No. 0406473), can be
employed according to the present disclosure to affect a fast
solution-phase immunochemical reaction. In these procedures, an
immobilizable immune complex is separated from the rest of the reaction
mixture by ionic interactions between the negatively charged
polyanion/immune complex and the previously treated, positively charged
matrix and detected by using any of a number of signal-generating
systems, including, e.g., chemiluminescent systems, as described in U.S.
application Ser. No. 921,979, corresponding to EPO Publication No. 0
273,115.

[0118] If the solid phase is silicon or glass, the surface must generally
be activated prior to attaching the capture antibody. Activated silane
compounds such as triethoxy amino propyl silane (available from Sigma
Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (Aldrich Chemical
Co., Milwaukee, Wis.), and (3-mercapto-propyl)-trimethoxy silane (Sigma
Chemical Co., St. Louis, Mo.) can be used to introduce reactive groups
such as amino-, vinyl, and thiol, respectively. Such activated surfaces
can be used to link the capture directly (in the cases of amino or
thiol), or the activated surface can be further reacted with linkers such
as glutaraldehyde, bis(succinimidyl) suberate, SPPD 9 succinimidyl
3-[2-pyridyldithio]propionate), SMCC
(succinimidyl-4-[Nmaleimidomethyl]cyclohexane-1-carboxylate), SlAB
(succinimidyl[4iodoacetyl]aminobenzoate), and SMPB (succinimidyl 4-[1
maleimidophenyl]butyrate) to separate the capture antibody from the
surface. Vinyl groups can be oxidized to provide a means for covalent
attachment. Vinyl groups can also be used as an anchor for the
polymerization of various polymers such as poly-acrylic acid, which can
provide multiple attachment points for specific capture antibodies. Amino
groups can be reacted with oxidized dextrans of various molecular weights
to provide hydrophilic linkers of different size and capacity. Examples
of oxidizable dextrans include Dextran T-40 (molecular weight 40,000
daltons), Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500
(molecular weight 500,000 daltons), Dextran T-2M (molecular weight
2,000,000 daltons) (all of which are available from Pharmacia,
Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons; available
from Sigma Chemical Co., St. Louis, Mo.). Additionally, polyelectrolyte
interactions can be used to immobilize a specific capture antibody on a
solid phase using techniques and chemistries described U.S. application
Ser. No. 150,278, filed Jan. 29, 1988, and U.S. application Ser. No.
375,029, filed Jul. 7, 1989, each of which is incorporated herein by
reference.

[0119] Other considerations affecting the choice of solid phase include
the ability to minimize non-specific binding of labeled entities and
compatibility with the labeling system employed. For, example, solid
phases used with fluorescent labels should have sufficiently low
background fluorescence to allow signal detection.

[0120] Following attachment of a specific capture antibody, the surface of
the solid support may be further treated with materials such as serum,
proteins, or other blocking agents to minimize non-specific binding
and/or to promote binding of autoantibodies.

Detection Systems in General

[0121] As discussed above, immunoassays according to the present
disclosure employ a second, detection antibody that is analyte-specific.
In certain embodiments, the second antibody has a detectable label.

[0122] Detectable labels suitable for use in the detection antibodies of
the present disclosure include any compound or composition having a
moiety that is detectable by spectroscopic, photochemical, biochemical,
immunochemical, electrical, optical, or chemical means. Such labels
include, for example, an enzyme, oligonucleotide, nanoparticle
chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence
quencher, or biotin. Thus for example, in an immunoassay employing an
optical signal, the optical signal is measured as an analyte
concentration dependent change in chemiluminescence, fluorescence,
phosphorescence, electrochemiluminescence, ultraviolet absorption,
visible absorption, infrared absorption, refraction, surface plasmon
resonance. In an immunoassay employing an electrical signal, the
electrical signal is measured as an analyte concentration dependent
change in current, resistance, potential, mass to charge ratio, or ion
count. In an immunoassay employing a change-of-state signal, the change
of state signal is measured as an analyte concentration dependent change
in size, solubility, mass, or resonance.

[0124] The label can be attached to the detection antibody prior to, or
during, or after contact with the biological sample. So-called "direct
labels" are detectable labels that are directly attached to or
incorporated into the detection antibody prior to use in the assay.
Direct labels can be attached to or incorporated into the detection
antibody by any of a number of means well known to those of skill in the
art.

[0125] In contrast, so-called "indirect labels" typically bind to the
detection antibody at some point during the assay. Often, the indirect
label binds to a moiety that is attached to or incorporated into the
detection agent prior to use. Thus, for example, a detection antibody can
be biotinylated before use in an assay. During the assay, an
avidin-conjugated fluorophore can bind the biotin-bearing detection
agent, to provide a label that is easily detected.

[0126] In another example of indirect labeling, polypeptides capable of
specifically binding immunoglobulin constant regions, such as polypeptide
A or polypeptide G, can also be used as labels for detection antibodies.
These polypeptides are normal constituents of the cell walls of
streptococcal bacteria. They exhibit a strong non-immunogenic reactivity
with immunoglobulin constant regions from a variety of species (see,
generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and
Akerstrom (1985) J. Immunol., 135: 2589-2542). Such polypeptides can thus
be labeled and added to the assay mixture, where they will bind to the
capture and detection antibodies, as well as to the autoantibodies,
labeling all and providing a composite signal attributable to analyte and
autoantibody present in the sample.

[0127] Some labels useful in the present disclosure may require the use of
an additional reagent(s) to produce a detectable signal. In an ELISA, for
example, an enzyme label (e.g., beta-galactosidase) will require the
addition of a substrate (e.g., X-gal) to produce a detectable signal. In
immunoassays using an acridinium compound as the direct label, a basic
solution and a source of hydrogen peroxide are added.

Detection Systems--Exemplary Formats

[0128] Chemiluminescence Immunoassay: In an exemplary embodiment, a
chemiluminescent compound is used in the above-described methods as a
direct label conjugated to the second, detection antibody. The
chemiluminescent compound can be an acridinium compound. When an
acridinium compound is used as the detectable label, then the
above-described method may further include generating or providing a
source of hydrogen peroxide to the mixture resulting from contacting the
test sample with the first antibody and the second antibody, and adding
at least one basic solution to the mixture to generate a light signal.
The light signal generated or emitted by the mixture is then measured to
detect the analyte of interest in the test sample.

[0129] The source of hydrogen peroxide may be a buffer solution or a
solution containing hydrogen peroxide or an enzyme that generates
hydrogen peroxide when added to the test sample. The basic solution
serves as a trigger solution, and the order in which the at least one
basic solution and detectable label are added is not critical. The basic
solution used in the method is a solution that contains at least one base
and that has a pH greater than or equal to 10, preferably, greater than
or equal to 12. Examples of basic solutions include, but are not limited
to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium
hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate,
calcium hydroxide, calcium carbonate and calcium bicarbonate. The amount
of basic solution added to the test sample depends on the concentration
of the basic solution used in the assay. Based on the concentration of
the basic solution used, one skilled in the art could easily determine
the amount of basic solution to be used in the method described herein.

[0130] In a chemiluminescence immunoassay according to the present
disclosure and using an acridinium compound as the detectable label,
preferably the acridinium compound is an acridinium-9-carboxamide.
Specifically, the acridinium-9-carboxamide has a structure according to
formula I:

##STR00005##

[0131] wherein R1 and R2 are each independently selected from
the group consisting of: alkyl, alkenyl, alkynyl, aryl or aralkyl,
sulfoalkyl, carboxyalkyl and oxoalkyl, and

[0132] wherein R3 through R15 are each independently selected
from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl or
aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen,
halide, nitro, cyano, sulfo, sulfoalkyl, carboxyalkyl and oxoalkyl; and
further wherein any of the alkyl, alkenyl, alkynyl, aryl or aralkyl may
contain one or more heteroatoms; and

[0141] Optionally, the test sample may be treated prior to the addition of
any one or more of the at least one basic solution, hydrogen peroxide
source and detectable label. Such treatment may include dilution,
ultrafiltration, extraction, precipitation, dialysis, chromatography and
digestion. Such treatment may be in addition to and separate from any
pretreatment that the test sample may receive or be subjected to as
discussed previously herein. Moreover, if such treatment methods are
employed with respect to the test sample, such treatment methods are such
that the analyte of interest remains in the test sample at a
concentration proportional to that in an untreated test sample (e.g.,
namely, a test sample that is not subjected to any such treatment
method(s)).

[0142] As mentioned briefly previously herein, the time and order in which
the test sample, the at least one basic solution, source of hydrogen
peroxide and the detectable label are added to form a mixture is not
critical. Additionally, the mixture formed by the at least one basic
solution, hydrogen peroxide source and the detectable label, can
optionally be allowed to incubate for a period of time. For example, the
mixture can be allowed to incubate for a period of time of from about 1
second to about 60 minutes. Specifically, the mixture can be allowed to
incubate for a period of from about 1 second to about 18 minutes.

[0143] When a chemiluminescent detectable label is used, after the
addition of the at least one basic solution, hydrogen peroxide source,
and the detectable label to the test sample, a detectable signal, namely,
a chemiluminescent signal, is generated. The signal generated by the
mixture is detected for a fixed duration of time. Preferably, the mixture
is formed and the signal is detected concurrently. The duration of the
detection may range from about 0.01 to about 360 seconds, more preferably
from about 0.1 to about 30 seconds, and most preferably from about 0.5 to
about 5 seconds. Chemiluminescent signals generated can be detected using
routine techniques known to those skilled in the art.

[0144] Thus, in a chemiluminescent immunoassay according to the present
disclosure, a chemiluminescent detectable label is used and added to the
test sample, the chemiluminescent signal generated after the addition of
the basic solution and the detectable label indicates the presence of the
analyte of interest in the test sample, which signal can be detected. The
amount or concentration of the analyte of interest in the test sample can
be quantified based on the intensity of the signal generated.
Specifically, the amount of the analyte of interest contained in a test
sample is proportional to the intensity of the signal generated.
Specifically, the amount of the analyte of interest present can be
quantified based on comparing the amount of light generated to a standard
curve for the analyte of interest or by comparison to a reference
standard. The standard curve can be generated using serial dilutions or
solutions to the analyte of interest of known concentration, by mass
spectroscopy, gravimetrically and by other techniques known in the art.

[0145] Fluorescence Polarization Immunoassay (FPIA): In an exemplary
embodiment, a fluorescent label is employed in a fluorescence
polarization immunoassay (FPIA) according to the invention. Generally,
fluorescent polarization techniques are based on the principle that a
fluorescent label, when excited by plane-polarized light of a
characteristic wavelength, will emit light at another characteristic
wavelength (i.e., fluorescence) that retains a degree of the polarization
relative to the incident light that is inversely related to the rate of
rotation of the label in a given medium. As a consequence of this
property, a label with constrained rotation, such as one bound to another
solution component with a relatively lower rate of rotation, will retain
a relatively greater degree of polarization of emitted light than when
free in solution.

[0146] This technique can be employed in immunoassays according to the
invention, for example, by selecting reagents such that binding of the
fluorescently labeled entities forms a complex sufficiently different in
size such that a change in the intensity light emitted in a given plane
can be detected. For example, when a labeled cardiac troponin antibody is
bound by one or more cardiac troponin antigens captured by the capture
antibody and/or autoantibodies reactive with the cardiac troponin, the
resulting complex is sufficiently larger, and its rotation is
sufficiently constrained, relative to the free labeled cardiac troponin
antibody that binding is easily detected.

[0147] Fluorophores useful in FPIA include fluorescein, amino fluorescein,
carboxyfluorescein, and the like, preferably 5 and
6-aminomethylfluorescein, 5 and 6-aminofluorescein, 6-carboxyfluorescein,
5-carboxyfluorescein, thioureafluorescein, and methoxytriazinolyl-amino
fluorescein, and similar fluorescent derivatives. Examples of
commercially available automated instruments with which fluorescence
polarization assays can be conducted include: the IMx system, the TDx
system, and TDxFLx system (all available from Abbott Laboratories, Abbott
Park, Ill.).

[0148] Scanning Probe Microscopy (SPM): The use of scanning probe
microscopy (SPM) for immunoassays also is a technology to which the
immunoassay methods of the present disclosure are easily adaptable. In
SPM, in particular in atomic force microscopy, the capture antibody is
affixed to the solid phase that in addition to being capable of binding
autoantibodies, has a surface suitable for scanning. The capture antibody
can, for example, be adsorbed to a plastic or metal surface.
Alternatively, the capture antibody can be covalently attached to, e.g.,
derivatized plastic, metal, silicon, or glass according to methods known
to those of ordinary skill in the art. Following attachment of the
capture antibody, the test sample is contacted with the solid phase, and
a scanning probe microscope is used to detect and quantify solid
phase-affixed complexes. The use of SPM eliminates the need for labels
that are typically employed in immunoassay systems. Such a system is
described in U.S. application Ser. No. 662,147, which is incorporated
herein by reference.

[0149] MicroElectroMechanical Systems (MEMS): Immunoassays according to
the present disclosure can also be carried out using a
MicroElectroMechanical System (MEMS). MEMS are microscopic structures
integrated onto silicon that combine mechanical, optical, and fluidic
elements with electronics, allowing convenient detection of an analyte of
interest. An exemplary MEMS device suitable for use in the present
disclosure is the Protiveris' multicantilever array. This array is based
on chemo-mechanical actuation of specially designed silicon
microcantilevers and subsequent optical detection of the microcantilever
deflections. When coated on one side with a binding partner, a
microcantilever will bend when it is exposed to a solution containing the
complementary molecule. This bending is caused by the change in the
surface energy due to the binding event. Optical detection of the degree
of bending (deflection) allows measurement of the amount of complementary
molecule bound to the microcantilever.

[0150] Electrochemical Detection Systems: In other embodiments,
immunoassays according to the present disclosure are carried out using
electrochemical detection, the techniques for which are well known to
those skilled in the art. Such electrochemical detection often employs
one or more electrodes connected to a device that measures and records an
electrical current. Such techniques can be realized in a number of
commercially available devices, such as the I-STAT® (Abbott
Laboratories, Abbott Park, Ill.) system, which comprises a hand-held
electrochemical detection instrument and self-contained assay-specific
reagent cartridges. For example, in the present invention, the basic
trigger solution could be contained in the self-contained hemoglobin
reagent cartridge and upon addition of the test sample, a current would
be generated at least one electrode that is proportional to the amount of
hemoglobin in the test sample. A basic procedure for electrochemical
detection has been described for example by Heineman and coworkers. This
entailed immobilization of a primary antibody (Ab, rat-anti mouse IgG),
followed by exposure to a sequence of solutions containing the antigen
(Ag, mouse IgG), the secondary antibody conjugated to an enzyme label
(AP-Ab, rat anti mouse IgG and alkaline phosphatase), and p-aminophenyl
phosphate (PAPP). The AP converts PAPP to p-aminophenol (PAPR, the
"R" is intended to distinguish the reduced form from the oxidized form,
PAPO, the quinoneimine), which is electrochemically reversible at
potentials that do not interfere with reduction of oxygen and water at pH
9.0, where AP exhibits optimum activity. PAPR does not cause
electrode fouling, unlike phenol whose precursor, phenylphosphate, is
often used as the enzyme substrate. Although PAPR undergoes air and
light oxidation, these are easily prevented on small scales and short
time frames. Picomole detection limits for PAPR and femtogram
detection limits for IgG achieved in microelectrochemical immunoassays
using PAPP volumes ranging from 20 μl to 360 μL, have been reported
previously. In capillary immunoassays with electrochemical detection, the
lowest detection limit reported thus far is 3000 molecules of mouse IgG
using a volume of 70 μl. and a 30 min or 25 min assay time.

[0151] In an exemplary embodiment employing electrochemical detection
according to the present disclosure, a capture antibody reactive with the
analyte of interest can be immobilized on the surface of an electrode
which is the solid phase. The electrode is then contacted with a test
sample from, e.g., a human. Any analyte in the sample binds to the
capture antibody to form a first solid phase-affixed complex.
Autoantibodies also bind to the surface of the electrode thereby becoming
immobilized on the surface of the electrode. Analyte in the test sample
that is unbound by the capture antibody binds to immobilized
autoantibodies that are reactive with the analyte to form a second solid
phase-affixed complex. These solid phase-affixed complexes are contacted
with a detection antibody that is analyte-specific and has a detectable
label. Formation of an immunodetection complex including the first
antibody-analyte-second antibody complex plus the
autoantibody-analyte-second antibody complex results in generation of a
signal by the detectable label, which is then detected.

[0163] The present disclosure also provides kits for assaying test samples
for presence of an analyte of interest wherein the test sample may
contain autoantibodies. Kits according to the present disclosure include
one or more reagents useful for practicing one or more immunoassays
according to the present disclosure. A kit generally includes a package
with one or more containers holding the reagents, as one or more separate
compositions or, optionally, as admixture where the compatibility of the
reagents will allow. The test kit can also include other material(s),
which may be desirable from a user standpoint, such as a buffer(s), a
diluent(s), a standard(s), and/or any other material useful in sample
processing, washing, or conducting any other step of the assay.

[0164] In certain embodiments, a test kit includes a humanized monoclonal
antibody, wherein the humanized monoclonal antibody is specific for the
analyte of interest. This component can be used as a positive control in
immunoassays according to the invention. If desired, this component can
be included in the test kit in multiple concentrations to facilitate the
generation of a standard curve to which the signal detected in the test
sample can be compared. Alternatively, a standard curve can be generated
by preparing dilutions of a single humanized monoclonal antibody solution
provided in the kit.

[0165] Kits according to the present disclosure can include a solid phase
capable of binding autoantibodies present in the test sample, a first
antibody that binds to at least one epitope on the analyte of interest,
the first antibody bound to the solid phase, a second antibody that binds
to at least one epitope on the analyte of interest, and instructions for
detecting or quantifying the analyte of interest. In certain embodiments
test kits according to the present disclosure may include the solid phase
as a material such as a magnetic particle, a bead, a test tube, a
microtiter plate, a cuvette, a membrane, a scaffolding molecule, a quartz
crystal, a film, a filter paper, a disc or a chip.

[0166] Test kits according to the present disclosure can include for
example non-human monoclonal antibodies against the analyte of interest,
as the first and second antibodies. The kit may also include a detectable
label that can be or is conjugated to the second antibody. In certain
embodiments, the test kit includes at least one direct label, which may
be an enzyme, oligonucleotide, nanoparticle chemiluminophore,
fluorophore, fluorescence quencher, chemiluminescence quencher, or
biotin. In some embodiments, the direct label is an acridinium compound
such as an acridinium-9-carboxamide according to formula I:

[0174] Test kits according to the present disclosure and which include an
acridinium compound can also include a basic solution. For example, the
basic solution can be a solution having a pH of at least about 10.

[0176] In certain embodiments, test kits according to the present
disclosure are configured for detection or quantification of one of the
following specific analytes of interest cardiac troponin, thyroid
stimulating hormone (TSH), beta human chorionic gonadotropin (beta-HCG);
myeloperoxidase (MPO), prostate specific antigen (PSA), human B-type
natriuretic peptide (BNP), myosin light chain 2, myosin-6 and myosin-7.
In such embodiments, the test kits include a first antibody and a second
antibody that each bind to an epitope on the selected analyte of
interest, i.e. a first antibody and a second antibody and second antibody
that each bind to an epitope on one of the following: cardiac troponin,
thyroid stimulating hormone (TSH), beta human chorionic gonadotropin
(beta-HCG); myeloperoxidase (MPO), prostate specific antigen (PSA), human
B-type natriuretic peptide (BNP), myosin light chain 2, myosin-6 and
myosin-7.

[0177] Test kits according to the present disclosure preferably include
instructions for carrying out one or more of the immunoassays of the
invention. Instructions included in kits of the present disclosure can be
affixed to packaging material or can be included as a package insert.
While the instructions are typically written or printed materials they
are not limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this disclosure.
Such media include, but are not limited to, electronic storage media
(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD
ROM), and the like. As used herein, the term "instructions" can include
the address of an internet site that provides the instructions.

D. ADAPTATIONS OF THE METHODS OF THE PRESENT DISCLOSURE

[0178] The present disclosure is for example applicable to the jointly
owned commercial Abbott Point of Care (1-STAT®) electrochemical
immunoassay system which performs sandwich immunoassays for several
cardiac markers, including TnI, CKMB and BNP. Immunosensors and ways of
operating them in single-use test devices are described in jointly owned
Publication Nos. US 20030170881, US 20040018577, US 20050054078, and US
20060160164, each of which is incorporated herein by reference.
Additional background on the manufacture of electrochemical and other
types of immunosensors is found in jointly owned U.S. Pat. No. 5,063,081
which is also incorporated by reference.

By way of example, and not of limitation, examples of the present
disclosures shall now be given.

[0183] Protocol: The human IgG standard solutions were assayed in
duplicate on an ARCHITECT i2000SR using the ARCHITECT stat-Troponin-I
protocol, and the microparticles and detection conjugate described above.
A point-to-point dose-response curve was constructed by plotting the RLU
response obtained from the ARCHITECT assay versus the human IgG
concentration tested as shown graphically in FIG. 3.

Example 2

Assay for Human IgG in Normal Serum on a Magnetic Microparticle Conjugated
to an Analyte Specific IgG. (ELN ref: E000777-281)

[0184] A normal serum sample was serially diluted in PBS and analyzed
using the reagents and protocol from Example 1. The RLU response from the
assay was plotted versus the human IgG concentration calculated from the
dose-response curve in Example 1 as shown graphically in FIG. 4.

[0185] Two samples were chosen from a population of normal blood donors
screened for anti-cardiac troponin-I autoantibodies (U.S. Ser. No.
11/588,073); one was determined to have low-reactivity (LR) in the assay
for while the other had high reactivity (HR). Cardiac troponin-I
(BiosPacific cat#J34170359) was added to aliquots of each sample at two
concentrations to give final cTnI concentrations of 0.25 and 1.5 ng/mL.
Each sample was analyzed using the microparticles described in Example 1
and cardiac troponin-I specific detection conjugate and diluents supplied
in the ARCHITECT Stat Troponin-I Kit (cat#2K41-30). The sample containing
a high level of autoantibodies reactive with cardiac troponin-I showed an
increased sensitivity to cardiac troponin-I at both the 0.25 and 1.5
ng/mL level. This is reflected in a 36-37% increase in the B/A and C/A
ratios in the HR sample relative to the LR samples, which had only a very
low level of autoantibodies reactive with cardiac troponin-I (Table 2).

[0186] One skilled in the art would readily appreciate that the
immunoassays described in the present disclosure are well adapted to
carry out the objects and obtain the ends and advantages mentioned, as
well as those inherent therein. The molecular complexes and the methods,
procedures, treatments, molecules, specific compounds described herein
are presently representative of preferred embodiments, are exemplary, and
are not intended as limitations on the scope of the invention. It will be
readily apparent to one skilled in the art that varying substitutions and
modifications may be made to the present disclosure disclosed herein
without departing from the scope and spirit of the invention.

[0187] All patents and publications mentioned in the specification are
indicative of the levels of those skilled in the art to which the present
disclosure pertains. All patents and publications are herein incorporated
by reference to the same extent as if each individual publication was
specifically and individually indicated to be incorporated by reference.

[0188] The present disclosure illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation or
limitations which are not specifically disclosed herein. Thus, for
example, in each instance herein any of the terms "comprising,"
"consisting essentially of" and "consisting of" may be replaced with
either of the other two terms. The terms and expressions which have been
employed are used as terms of description and not of limitation, and
there is no intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are possible
within the scope of the present disclosure claimed. Thus, it should be
understood that although the present disclosure has been specifically
disclosed by preferred embodiments and optional features, modification
and variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by the
appended claims.